Date of Award
Restricted Access Dissertation
Doctor of Philosophy
Graduate School of Biomedical Sciences
S. Dan Dimitrijevich
These studies evaluated the possibility of inducing the cells of human neonatal neurospheres to commit to the neuronal phenotype. Neurospheres are hollow, multicellular aggregates held together by combinations of adherens junctions and gap junctions. Their walls were seen to be 6-8 cell layers thick, with proliferating cells randomly distributed throughout these layers. The cells of neurosphere wall were found to be organized into an outer “glial basket” and an inner layer of putative neuroblasts, and this arrangement facilitated orchestrated cellular outgrowth on immobilized extracellular matrix proteins, with GFAP+/nestin- cells forming radial tracks upon which GFAp=/nestin+ cells migrated. Using a novel technique, it was demonstrated that FGF2 downregulated nestin and vimentin, induced transient upregulations of α-internexin, and induced sustained upregulations of neurofilament M (NF-M). β-tubulin was most strongly upregulated by long-term (9 days) exposure to basal medium without growth factors. Dose response studies indicated that 5ng/ml FGF2 was optimal for promoting upregulations of the neuronal intermediate filament proteins, but that 0-1ng/ml FGF2 was optimal for promoting upregulations of the neuronal intermediate filament proteins, but that 0-1ng/ml FGF2 was optimal for β-tubulin upregulation. Commitment-promoting FGF2 treatments were shown to have little effect on the proliferation of the neurosphere cells with the exception of treatment with growth factor-free basal medium, which strongly reduced proliferation. The α, βI, βII, δ, ε, η, and θ isoforms of PKC were detected in neurospheres, and these expression profiles were quantitatively but not qualitatively altered by treatments with various growth factors. Blockade of PKC activity by administration of the general PKC inhibitor GF109203X ablated FGF2-induced upregulations of α-internexin and NF-M, although FGF2 and GF109203X upregulated the expression of β-tubulin. We propose a model in which high FGF2 coupled with EGF drives cellular proliferation, the removal of EGF and decreased FGF2 stimulates upregulation of neuronal intermediate filaments, and a further lowering of FGF2 (down to 0ng/ml) stimulates the upregulation of β-tubulin and axonal extension. During the first two stages, cellular proliferation is not altered, and it is not until the final stage that cells begin to exit the cell cycle. It is presumed that PKC drives the first two stages, while the final stage is inhibited by PKC.
Moeller, M. L.
"Induction of Neuronal Commitment in Pluripotent Neurospheres" Fort Worth, Tx: University of North Texas Health Science Center;